Photosystem II (PSII) is a large, photosynthetic membrane protein complex responsible for water oxidation and the formation of oxygen on Earth. Understanding the structure of this protein is important for studying photosynthetic energy transfer and assembly mechanisms in higher plants. Recently, technological breakthroughs in the field of cryo-EM have lead to the ability to study this membrane protein by single particle analysis (SPA). This technique does not require the growth of crystals, a challenge that has hindered structural determination in higher plant PSII. In this work, the biochemical preparation of spinach PSII for cryo-EM is optimized to maintain high activity. Subunit characterization, oxygen activity, UV absorbance spectroscopy, mass spectrometry, and negative stain transmission electron microscopy were employed to characterize the spinach PSII preparation. Cryo-EM experiments resulted in a first model of a highly active C2 PSII complex, which contains all intrinsic and extrinsic subunits essential for activity.